Population structure and attribution of human clinical Campylobacter jejuni isolates from central Europe to livestock and environmental sources.

Campylobacter jejuni is among the most prevalent causes of human bacterial gastroenteritis worldwide. Domesticated animals and, especially, chicken meat are considered to be the main sources of infections. However, the contribution of surface waters and wildlife in C. jejuni transmission to humans is not well understood. We have evaluated the source attribution potential of a six-gene multiplex PCR (mPCR) method coupled with STRUCTURE analysis on a set of 410 C. jejuni strains isolated from environment, livestock, food and humans in central Europe. Multiplex PCR fingerprints were analysed using Subclade prediction algorithm to classify them into six distinct mPCR clades. A subset of C. jejuni isolates (70%) was characterized by multilocus sequence typing (MLST) demonstrating 74% congruence between mPCR and MLST. The correspondence analysis of mPCR clades and sources of isolation indicated three distinct groups in the studied C. jejuni population-the first one associated with isolates from poultry, the second one with isolates from cattle, and the third one with isolates from the environment. The STRUCTURE analysis attributed 7.2% and 21.7% of human isolates to environmental sources based on MLST and mPCR fingerprints, respectively.

Combined Campylobacter jejuni and Campylobacter coli Rapid Testing and Molecular Epidemiology in Conventional Broiler Flocks.

Campylobacter spp. are important causes of bacterial zoonosis, most often transmitted by contaminated poultry meat. From an epidemiological and risk assessment perspective, further knowledge should be obtained on Campylobacter prevalence and genotype distribution in primary production. Consequently, 15 Austrian broiler flocks were surveyed in summer for their thermophilic Campylobacter spp. contamination status. Chicken droppings, dust and drinking water samples were collected from each flock at three separate sampling periods. Isolates were confirmed by PCR and subtyped. We also compared three alternative methods (culture-based enrichment in Bolton broth, culture-independent real-time PCR and a lateral-flow test) for their applicability in chicken droppings. Twelve flocks were found to be positive for thermophilic Campylobacter spp. during the entire sampling period. Seven flocks (46.6%) were contaminated with both, C. jejuni and C. coli, five flocks harboured solely one species. We observed to a majority flock-specific C. jejuni and C. coli genotypes, which dominated the respective flock. Flocks within a distance <2 km shared the same C. jejuni genotypes indicating a cross-contamination event via the environment or personnel vectors. Multilocus sequence typing (MLST) of C. jejuni revealed that the majority of isolates were assigned to globally distributed clonal complexes or had a strong link to the human interface (CC ST-446 and ST4373). The combination of techniques poses an advantage over risk assessment studies based on cultures alone, as, in the case of Campylobacter, occurrence of a high variety of genotypes might be present among a broiler flock. We suggest applying the lateral-flow test under field conditions to identify 'high-shedding' broiler flocks at the farm level. Consequently, poultry farmers and veterinarians could improve hygiene measurements and direct sanitation activities, especially during the thinning period. Ultimately, real-time PCR could be applied to quantify Campylobacter spp. directly from chicken droppings and avoid non-interpretable results achieved by culture-dependent methods.

Urban prevalence of Listeria spp. and Listeria monocytogenes in public lavatories and on shoe soles of facility patrons in the European capital city Vienna.

The aim of this study was to determine the prevalence of Listeria spp. and Listeria monocytogenes (L. monocytogenes) in urban public lavatories and on shoe soles of facility patrons in a European capital city. More than 91% of all municipal public lavatories in Vienna close to public hubs were included in this study. Overall, 373 swab samples of public lavatories and shoes of facility patrons were enriched, according to ISO 11290-1. Listeria monocytogenes isolates were subtyped using pulsed-field gel electrophoresis. A total of 24 samples were positive for Listeria spp., yielding an overall prevalence of 6.4% (24/373). Listeria monocytogenes was found in 2.1% (8/373) of all samples. Swabs from lavatories in parks, container lavatories and lavatories at markets had the highest prevalences of 20.7% (6/29), 20% (2/10) and 12.5% (1/8) Listeria spp., respectively. These detection rates were statistically significantly higher than those associated with lavatories in shopping centres (P = 0.003, P = 0.002, P = 0.02) and at public transport locations (P = 0.0004, P = 0.005, P = 0.02). Shoes sampled at Christmas markets showed the highest Listeria spp. and L. monocytogenes prevalences of 80% (4/5) and 40% (2/5), respectively. With regard to shoe type, Listeria spp. detection rates were 14.3% (3/21; winter boots), 13.3% (2/15; hiking boots), sport shoes (5.9%; 2/34) and brogues (5.1%; 4/79). No Listeria spp. were found on shoe soles that had smooth treads (0/76), while Listeria spp. were detected on 19.5% (8/41) of medium depth tread shoe types and on 9.4% (3/32) of deep tread shoes. These data suggest that soil environment is still one of the most important reservoirs for the foodborne pathogen L. monocytogenes.

L. monocytogenes in a cheese processing facility: Learning from contamination scenarios over three years of sampling.

The aim of this study was to analyze the changing patterns of Listeria monocytogenes contamination in a cheese processing facility manufacturing a wide range of ready-to-eat products. Characterization of L. monocytogenes isolates included genotyping by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Disinfectant-susceptibility tests and the assessment of L. monocytogenes survival in fresh cheese were also conducted. During the sampling period between 2010 and 2013, a total of 1284 environmental samples were investigated. Overall occurrence rates of Listeria spp. and L. monocytogenes were 21.9% and 19.5%, respectively. Identical L. monocytogenes genotypes were found in the food processing environment (FPE), raw materials and in products. Interventions after the sampling events changed contamination scenarios substantially. The high diversity of globally, widely distributed L. monocytogenes genotypes was reduced by identifying the major sources of contamination. Although susceptible to a broad range of disinfectants and cleaners, one dominant L. monocytogenes sequence type (ST) 5 could not be eradicated from drains and floors. Significantly, intense humidity and steam could be observed in all rooms and water residues were visible on floors due to increased cleaning strategies. This could explain the high L. monocytogenes contamination of the FPE (drains, shoes and floors) throughout the study (15.8%). The outcome of a challenge experiment in fresh cheese showed that L. monocytogenes could survive after 14days of storage at insufficient cooling temperatures (8 and 16°C). All efforts to reduce L. monocytogenes environmental contamination eventually led to a transition from dynamic to stable contamination scenarios. Consequently, implementation of systematic environmental monitoring via in-house systems should either aim for total avoidance of FPE colonization, or emphasize a first reduction of L. monocytogenes to sites where contamination of the processed product is unlikely. Drying of surfaces after cleaning is highly recommended to facilitate the L. monocytogenes eradication.

Population diversity of Listeria monocytogenes in quargel (acid curd cheese) lots recalled during the multinational listeriosis outbreak 2009/2010.

It has been possible to determine the genotype diversity of Listeria monocytogenes in the actual cheese lots of acid curd cheese that caused a multinational outbreak between 2009 and 2010. Following product recall in January 2010 all lots were investigated. A total of 422 L. monocytogenes isolates were characterized by genotyping. In a first approach the PCR serogroups were defined by multiplex-PCR assays. Subsequently, the isolates were subtyped by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Sequence types were assigned by submitting the DNA sequences to the Listeria MLST database at the Institute Pasteur. The serogroup PCR resulted in a homogeneous 1/2a - 3a (genetic linage II) cluster. The generated PFGE patterns divided the strains into two clusters (type 1 and 2) diverging at a homogeneity level of 74%. PFGE-type 2 was predominant, accounting for 98.3% (n = 415/422) of the isolates and was isolated during the whole period of acid curd cheese processing (01.12.2009-13.01.2010). 1.7% of all tested L. monocytogenes isolates (n = 7/422) belonged to PFGE-type 1 and were isolated from 28% of all cheese lots (n = 5/18) produced between the time span of 08.12.2009 to 13.01.2010. Furthermore, PFGE-type 1 and 2 showed the same PFGE patterns as the human outbreak strains (clone 1 and clone 2).

Performance testing of six chromogenic ALOA-type media for the detection of Listeria monocytogenes.

AIMS: The aim of the study was to test the performance of commercially available chromogenic plating media for detection and enumeration of the food-borne pathogen Listeria monocytogenes. A wide range of chromogenic media similar to Agar Listeria according to Ottaviani and Agosti (ALOA) were compared using PALCAM agar, according to van Netten et al. METHODS AND RESULTS: Six chromogenic media similar to ALOA were challenged for inclusivity and exclusivity. Additionally, the ability of chromogenic agars to facilitate growth of stressed L. monocytogenes strains and mixed cultures with competitive non-Listeria strains was estimated. Finally, we tested the detection and enumeration of L. monocytogenes in artificially inoculated and naturally contaminated food samples. The results of this study indicated that chromogenic media are a good supplementation to PALCAM agar. A single application is not advisable, as the specificity of chromogenic agars is frequently insufficient (50.0-88.9%), particularly in food samples with a complex microflora. CONCLUSIONS: The competitive flora of food samples is able to overgrow low numbers of L. monocytogenes, especially in half-Fraser enrichment. This might lead to the underestimation of L. monocytogenes positive samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Although many evaluation studies of chromogenic agar have been published recently, harmonized validation strategies are lacking. This survey provides a new concept for stepwise testing of plating media.